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Abstract

:
A new cytotoxic 19-oxygenated steroid, nebrosteroid Q (1) and two new cytotoxic 19-norergosterols, nebrosteroids R and S (2 and 3) were isolated from the soft coral Nephthea chabrolii collected at San-Hsian-Tai. The structures of nebrosteroids Q–S (1–3) were elucidated by spectral analysis, and their cytotoxicity against selected cancer cells as well as antiviral activity against human cytomegalovirus (HCMV) were measured in vitro.

1. Introduction

Numerous secondary metabolites including steroids, sesquiterpenoids, diterpenoids, and meroditerpenoids have been isolated from soft corals of the genus Nephthea [1,2,3,4,5,6,7,8,9,10,11,12,13,14,15,16,17,18,19,20,21,22,23,24,25,26]. Previous studies on these materials showed them to exhibit diverse biological properties including cytotoxic [3,4,5,6,17,19,26], anti-inflammatory [12,13,22,25] and antimicrobial activities [18]. The acetone extract of the soft coral Nephthea chabrolii (Figure 1) was found to be cytotoxic towards P-388 mouse lymphocytic leukemia cells. Chromatographic fractionation led to the isolation of a new cytotoxic 19-oxygenated steroid, nebrosteroid Q (1) and two new cytotoxic 19-norergosterols, nebrosteroids R and S (2 and 3) (Figure 2).

Nebrosteroid R (2) was isolated as a white amorphous powder. HRESIMS of 2 exhibited a pseudo molecular ion peak at m/z 423.3241 [M + Na]+ (calcd for 423.3239) and established a molecular formula of C27H44O2, indicating six degrees of unsaturation. The 13C NMR (Table 1) displayed 27 carbon signals, which were identified by the assistance of the DEPT spectrum as four methyls, eleven methylenes, eight methines, and four quaternary carbons. The 1H NMR signal [δH 4.09 (m, 1H), 3.83 (brs, 1H)] (Table 1) and IR absorption at 3423 cm−1, together with the observation of two oxygen-bearing carbon resonances (δC 65.8 and 68.6) in the 13C NMR spectrum, revealed the presence of two hydroxyl groups. Furthermore, one tetrasubstituted double bond (δC 125.8 and 135.4), and one terminal double bond (δC 105.6 and 156.8) were assigned from 13C NMR and DEPT spectra of 2. The above functionalities accounted for two of the six degrees of unsaturation, suggesting a tetracyclic skeleton for 2. Interpretation of the 1H-1H COSY spectrum led to partial structures III and IV (Figure 3). The connectivities of these partial structures were further established by the HMBC correlations (Figure 3). Moreover, the COSY correlations from H2-1 to H-3 through H2-2 and from H-8 to H-6 through H2-7 led to the assignment of the secondary hydroxyl groups at C-3 and C-6. The location of the tetrasubstituted double bond at C-5/C-10 was clarified by analysis of the HMBC correlations from H2-6 to C-10, H2-2 to C-10, H-11 to C-10, and H-6 to C-5. The NOESY correlations (Figure 5) observed between H-3 and H-2α, H-3 and H-4α, H-4α and H-6α, H-6α and H-7α, H-7α and H-9, H-7β and H-8 indicated the β-orientations of the hydroxyl groups at C-3 and C-6. Moreover, the NOESY correlations observed between H-2β and H-1β, H-4α and H-6α, H-6α and H-7α, H-7α and H-9, H-7β and H-8, H-9 and H-14, H-11β and H-8, H-12β and Me-18, Me-18 and H-20, and Me-21 and H-12β in 2 confirmed the relative configurations for each ring junction and chiral center. Thus, the structure of 2 was established unambiguously.

Figure 5.
NOESY correlations of compound 2.

Figure 5.
NOESY correlations of compound 2.

Nebrosteroid R (3) was isolated as a white amorphous powder. HRESIMS of 3 exhibited a pseudo molecular ion peak at m/z 437.3398 [M + Na]+ (calcd for 437.3395) and established a molecular formula of C28H46O2, indicating six degrees of unsaturation. The 13C NMR (Table 1) displayed 28 carbon signals, which were identified with the assistance of the DEPT spectrum as five methyls, eleven methylenes, eight methines, and four quaternary carbons. The 1H NMR signal [δH 4.03 m (m, 1H)] (Table 1) and IR absorption at 3445 cm−1, together with the observation of one oxygen-bearing carbon resonance (δC 66.1) in the 13C NMR spectrum, revealed the presence of a secondary hydroxyl group. The 1H NMR signal [δH 3.31 (brs, 1H), 3.34 (s, 3H)] together with the observation of two oxygen-bearing carbon resonances (δC 57.0 and 78.1 in the 13C NMR spectrum, revealed the presence of a secondary methoxyl group. Furthermore, one tetrasubstituted double bond (δC 124.7 and 135.6), and one terminal double bond (δC 105.6 and 156.9) were assigned from 13C NMR and DEPT spectra of 3. The above functionalities accounted for two of the six degrees of unsaturation, suggesting a tetracyclic skeleton for 3. Interpretation of the 1H-1H COSY spectrum led to two similar partial structures as 2. The connectivities of these partial structures were further established by HMBC correlations as 2. Moreover, the COSY correlations from H2-1 to H-3 through H2-2 and from H-8 to H-6 through H2-7 as well as HMBC correlations from 6-OMe to H-6 led to the assignment of the secondary hydroxyl group at C-3 and the secondary methoxyl group at C-6. The location of the tetrasubstituted double bond at C-5/C-10 was clarified by analysis of the HMBC correlations from H2-6 to C-10, H2-2 to C-10, H-11 to C-10, and H-7 to C-5. The NOESY correlations observed between H-3 and H-2α, H-3 and H-4α, H-4α and H-6α, H-6α and H-7α, H-7α and H-9, H-7β and H-8 indicated the β-orientation of the hydroxyl group at C-3 and β-orientation of methoxyl group at C-6. Moreover, the NOESY correlations observed between H-2β and H-1β, H-4α and H-6α, H-6α and H-7α, H-7α and H-9, H-7β and H-8, H-9 and H-14, H-11β and H-12β, H-12β and Me-18, Me-18 and H-20, and Me-21 and H-12β in 3 confirmed the relative configurations for each ring junction and chiral center. Thus, the structure of 3 was established unambiguously.

3.2. Biological Material

The soft coral N. chabrolii was collected by hand using scuba off the San-Hsian-Tai coast, Taitong County, Taiwan, in July 2009 at a depth of 6 m and stored in a freezer until extraction. The voucher specimen (SST-32) was identified by Prof. Chang-Feng Dai, National Taiwan University and deposited at the Department of Marine Biotechnology and Resources, National Sun Yat-sen University, Taiwan.

3.3. Extraction and Isolation

A specimen of soft coral N. chabrolii (2.2 kg) was minced and extracted with acetone (3 × 4 L) at room temperature. The combined acetone extracts were then partitioned between H2O and EtOAc. The resulting EtOAc extract (24.6 g) was subjected to gravity silica gel 60 column chromatography (Si 60 CC) using n-hexane and n-hexane/EtOAc of increasing polarity, to give 22 fractions. The fraction 12 (0.84 g), eluted with n-hexane/EtOAc (1:10), was further subjected to Si 60 CC (n-hexane/EtOAc, 10:1 to 100% EtOAc) to give six subfractions. A subfraction 12-2 (299 mg), was separated by a RP-18 flash column (MeOH/H2O, 50:50 to 100% MeOH) to give eight fractions. The subfraction 12-2-6, eluted with MeOH/H2O (90:10), was purified by RP-18 HPLC (MeOH/H2O, 95:5) to afford 1 (2.5 mg). The fraction 13 (0.69 g), eluted with EtOAc, was further subjected to Si 60 CC (n-hexane/EtOAc, 50:1 to 100% EtOAc) to give four subfractions. The subfraction 13-3 (299 mg), was separated by a RP-18 flash column (MeOH/H2O, 45:55 to 100% MeOH) to give three fractions. In turn, a subfraction 13-3-3, eluted with MeOH/H2O (90:10), was further purified by RP-18 HPLC (MeOH/H2O, 95:5) to afford 2 (3.0 mg) and 3 (1.0 mg).

3.4. Cytotoxicity Assay

Cytotoxicity was determined on P-388 (mouse lymphocytic leukemia), HT-29 (human colon adenocarcinoma), and A-549 (human lung epithelial carcinoma) tumor cells using a modification of the MTT colorimetric method according to a previously described procedure [27,28]. The provision of the P-388 cell line was supported by J. M. Pezzuto, formerly of the Department of Medicinal Chemistry and Pharmacognosy, University of Illinois at Chicago. HT-29 and A-549 cell lines were purchased from the American Type Culture Collection. To measure the cytotoxic activities of tested compounds, five concentrations (50, 10, 2, 0.4, 0.08 μg/mL) with three replications were performed on each cell line. Mithramycin was used as a positive control.

3.5. Anti-HCMV Assay

To determine the effects of natural products upon HCMV cytopathic effect (CPE), confluent human embryonic lung (HEL) cells grown in 24-well plates were incubated for 1 h in the presence or absence of various concentrations of tested natural products with three replications. Ganciclovir was used as a positive control. Then, cells were infected with HCMV at an input of 1000 pfu (plaque forming units) per well of a 24-well dish. Antiviral activity was expressed as IC50 (50% inhibitory concentration), or compound concentration required to reduce virus induced CPE by 50% after seven days as compared with the untreated control. To monitor the cell growth upon treating with natural products, an MTT-colorimetric assay was employed [29].

4. Conclusion

This investigation of soft coral N. chabrolii collected at San-Hsian-Tai (Taitong County, Taiwan) has led to the isolation of a new cytotoxic 19-oxygenated steroid, nebrosteroid Q (1) and two new cytotoxic norergosterols, nebrosteroids R and S (2 and 3). Nebrosteroids Q–S (1–3) exhibited cytotoxicity against P-388 cell line with ED50 of 1.0, 1.2, and 1.0 μg/mL, respectively. However, previously isolated cholestene derivatives, nebrosteroids I–K [12] did not show cytotoxicity. In order to rule out the possibility of 3 being an isolation artifact, a solution of 2 was kept at room temperature for three days in the presence of Si-60 or RP-18 gel in MeOH. However, the formation of 3 was not observed.

Acknowledgments

This research was financially supported by grants from the National Science Council (NSC99-2628-B-110-002-MY3) and Ministry of Education of Taiwan awarded to C.-Y.D.